PROJECT SUMMARY ? Steitz Biochemical, molecular genetic and high resolution X-ray crystallographic experiments will be used to establish the structural bases for the functional mechanisms by which the ribosome and associated factors achieve their roles in protein synthesis. To further illuminate the mechanisms of each step in the process of protein synthesis, we wish to obtain crystals of the ribosome trapped in as many of the steps in the protein synthesis cycle as possible and to establish their structures at the highest resolution possible. The complexes whose structures will be pursued include those of the 1) 70S ribosome with bound m-RNA, tRNA and elongation factor G with a GTP analogue, back translocation factor LepA with a GTP analogue and a complex with IF-3, 70S ribosome complexes with rescue factors that rescue stalled ribosomes. To explore the differences between the bacterial ribosome and its much large counterpart from eukaryotes, particularly in the initiation step of protein synthesis, the structures of the 40S subunit, the 60S subunit and/or the 80S ribosome, and using ribosomes isolated from either yeast or rabbit reticulocyte will be pursued. The structure of the 70S ribosome bound to IF-2 will be determined and compared to the complex of the 40S subunit with bound eIF-2. 3) We shall also continue and expand our structural studies of the ribozyme Twister and Twister's Sister, as well as new ribozymes being discovered by the Breaker laboratory.